In macrophages, cellular iron-metabolism status is tightly integrated with macrophage phenotype. Although mitochondrial function regulates immune-cell phenotype and inflammatory responses, how molecular events regulate mitochondrial activity to integrate regulation of iron metabolism and macrophage phenotype remains unclear. In this study, we explored the important role of the actin-regulatory protein glia maturation factor-gamma (GMFG) in the regulation of cellular iron metabolism and macrophage phenotype. GMFG knockdown regulated the expression of iron-metabolism proteins and increased iron levels in murine macrophages, and concomitantly promoted their polarization towards an anti-inflammatory M2 phenotype. GMFG-knockdown macrophages exhibited moderately increased levels of mitochondrial reactive oxygen species (mtROS), which were accompanied by decreased expression of some mitochondrial respiration chain components, including the iron-sulfur cluster assembly scaffold protein ISCU, as well as of the anti-oxidant enzymes SOD1 and SOD2. Importantly, treatment of GMFG-knockdown macrophages with the anti-oxidant N-acetylcysteine (NAC) reversed the altered expression of iron-metabolism proteins and partially inhibited the enhanced gene expression of M2 macrophage markers, suggesting that mtROS are mechanistically linked to cellular iron metabolism and macrophage phenotype. Finally, GMFG interacted with the mitochondrial membrane protein ATPase family AAA domain-containing protein 3A (ATAD3A), and GMFG knockdown decreased expression of the endoplasmic reticulum (ER) protein GRP78, implying that GMFG knockdown-induced mtROS might be attributed to alteration of mitochondria-ER contacts in macrophages. Collectively, our findings suggest that GMFG is an important modulator in macrophages for regulating cellular iron metabolism and macrophage phenotype, and could be a novel therapeutic target for modulating macrophage function in immune and metabolic disorders.